Partial Magneto-Endosomalysis for Cytosolic Delivery of Antibodies.

Modulation of protein functions and interactions is the most direct and effective means to intervene in cellular processes and pathogenesis. The majority of the critical intracellular signaling pathways, however, are considered undruggable using small molecules. In this regard, antibodies are superior in structural and functional diversity and are significantly easier to raise compared to the screening of small molecules. Despite these advantages, the uses of antibodies in live cells (either as an imaging agent or as a therapeutic compound) are substantially undermined, only acting on extracellular targets. The inability of targeting intracellular proteins is because of a fundamental issue: antibodies enter cells through endocytosis where the vast majority are trapped in endosomes for degradation. Here, we report a nanoparticle self-assembly strategy enabling antibody endosomal escape. We demonstrate the intracellular bioavailability of antibodies and the preserved binding specificity to their cytosolic targets. This technology is simple and opens exciting opportunities for live-cell imaging, therapeutics development, and cell engineering.

Magneto-Endosomalytic Therapy for Cancer.

A remarkably simple yet effective mode of cancer treatment is reported by repurposing clinically approved magnetic nanoparticles (MNPs). Intracellular nanoparticle self-assembly directed by static parallel magnetic fields leads to cell death in targeted tissues while leaving other cells and organs intact. This simple concept opens a new avenue to treat cancer, capitalizing on nanosciences and the nanoparticle (NP) design principles accumulated in the past decades.

Cytosolic delivery of proteins by cholesterol tagging.

Protein-based imaging agents and therapeutics are superior in structural and functional diversity compared to small molecules and are much easier to design or screen. Antibodies or antibody fragments can be easily raised against virtually any target. Despite these fundamental advantages, the power and impact of protein-based agents are substantially undermined, only acting on a limited number of extracellular targets because macrobiomolecules cannot spontaneously cross the cell membrane. Conventional protein delivery techniques fail to address this fundamental problem in that protein cargos are predominantly delivered inside cells via endocytosis, a remarkably effective cell defense mechanism developed by Mother Nature to prevent intact biomolecules from entering the cytoplasm. Here, we report a unique concept, noncovalent cholesterol tagging, enabling virtually any compact proteins to permeate through the cell membrane, completely bypassing endocytosis. This simple plug-and-play platform greatly expands the biological target space and has the potential to transform basic biology studies and drug discovery.

Combining Qdot Nanotechnology and DNA Nanotechnology for Sensitive Single-Cell Imaging.

Immunohistochemistry (IHC) can provide detailed information about protein expression within the cell microenvironment and is one of the most common techniques in biology and medicine due to the broad availability of highly specific antibodies and well-established bioconjugation methods for modification of these antibodies with chromogens and fluorophores. Despite recent advances in this field, it remains challenging to simultaneously achieve high multiplexing, sensitivity, and throughput in single-cell profiling experiments. Here, the combination of two powerful technologies is reported, quantum dot and signal amplification by exchange reaction (QD-SABER), for sensitive and multiplexed imaging of endogenous proteins. Compared to the conventional IHC process using dye-labeled secondary antibodies (which already has a built-in signal amplification mechanism), QD-SABER provides an additional 7.6-fold signal amplification. In addition, the DNA hybridization-based IHC can be rapidly removed to regenerate the sample for subsequent cycles of immunostaining (>10 cycles), greatly expanding the multiplexing capability.

Triplex DNA Nanoswitch for pH-Sensitive Release of Multiple Cancer Drugs.

A DNA-based stimulus-responsive drug delivery system for synergetic cancer therapy has been developed. The system is built on a triplex-DNA nanoswitch capable of precisely responding to pH variations in the range of ∼5.0-7.0. In extracellular neutral pH space, the DNA nanoswitch keeps a linear conformation, immobilizing multiple therapeutics such as small molecules and antisense compounds simultaneously. Following targeted cancer cell uptake via endocytosis, the nanoswitch inside acidic intracellular compartments goes through a conformational change from linear to triplex, leading to smart release of the therapeutic combination. This stimuli-responsive drug delivery system does not rely on artificial responsive materials, making it biocompatible. Furthermore, it enables simultaneous delivery of multiple therapeutics for enhanced efficacy. Using tumor-bearing mouse models, we show efficient gene silencing and significant inhibition of tumor growth upon intravenous administration of the smart nanoswitch, providing opportunities for combinatorial cancer therapy.

Membrane-Penetrating Carbon Quantum Dots for Imaging Nucleic Acid Structures in Live Organisms.

The dynamics of DNA and RNA structures in live cells are important for understanding cell behaviors, such as transcription activity, protein expression, cell apoptosis, and hereditary disease, but are challenging to monitor in live organisms in real time. The difficulty is largely due to the lack of photostable imaging probes that can distinguish between DNA and RNA, and more importantly, are capable of crossing multiple membrane barriers ranging from the cell/organelle to the tissue/organ level. We report the discovery of a cationic carbon quantum dot (cQD) probe that emits spectrally distinguishable fluorescence upon binding with double-stranded DNA and single-stranded RNA in live cells, thereby enabling real-time monitoring of DNA and RNA localization and motion. A surprising finding is that the probe can penetrate through various types of biological barriers in vitro and in vivo. Combined with standard and super-resolution microscopy, photostable cQDs allow time-lapse imaging of chromatin and nucleoli during cell division and Caenorhabditis elegans (C. elegans) growth.

Quantum dots and mouse strain influence house dust mite-induced allergic airway disease.

Quantum dot nanoparticles (QDs) are engineered nanomaterials (ENMs) that have utility in many industries due to unique optical properties not available in small molecules or bulk materials. QD-induced acute lung inflammation and toxicity in rodent models raise concerns about potential human health risks. Recent studies have also shown that some ENMs can exacerbate allergic airway disease (AAD). In this study, C57BL/6J and A/J mice were exposed to saline, house dust mite (HDM), or a combination of HDM and QDs on day 1 of the sensitization protocol. Mice were then challenged on days 8, 9 and 10 with HDM or saline only. Significant differences in cellular and molecular markers of AAD induced by both HDM and HDM + QD were observed between C57BL/6J and A/J mice. Among A/J mice, HDM + QD co-exposure, but not HDM exposure alone, significantly increased levels of bronchoalveolar lavage fluid (BALF). IL-33 compared to saline controls. BALF total protein levels in both mouse strains were also only significantly increased by HDM + QD co-exposure. In addition, A/J mice had significantly more lung type 2 innate lymphoid cells (ILC2s) cells than C57BL/6J mice. A/J lung ILC2s were inversely correlated with lung glutathione and MHC-IIhigh resident macrophages, and positively correlated with MHC-IIlow resident macrophages. The results from this study suggest that 1) QDs influence HDM-induced AAD by potentiating and/or enhancing select cytokine production; 2) that genetic background modulates the impact of QDs on HDM sensitization; and 3) that potential ILC2 contributions to HDM induced AAD are also likely to be modulated by genetic background.

Scalable Production of Therapeutic Protein Nanoparticles Using Flash Nanoprecipitation.

Flash nanoprecipitation (FNP) by fast mixing of drug-containing organic solvent and water in a microchamber is a powerful and scalable technology to produce solid drug nanoparticles with high payload. The embedded therapeutic drugs, however, are largely limited to hydrophobic small molecules. By transferring proteins into organic solvent via hydrophobic ion pairing, the scope of FNP applications is expanded. This platform technology is capable of producing protein nanoparticles with tunable sizes (from ≈30 nm to sub-micrometers), high-production scale (grams per hour), high drug loading efficiency (>90%), and excellent reproducibility, opening a new paradigm for formulation of biological pharmaceuticals. As a proof-of-concept, insulin nanoparticles are made to address a major medical challenge; oral administration. A relative insulin bioavailability of 13.2% is achieved, enabling sustained reduction of blood glucose levels in a diabetic rat model.

Quantum dot induced acute changes in lung mechanics are mouse strain dependent.

INTRODUCTION: Concerns have been raised regarding occupational exposure to engineered nanomaterials (ENMs). Potential impacts on lung function from inhalation exposures are of concern as the lung is a sensitive ENM target in animals. Epidemiological data suggest that occupational exposure to ENMs may impact respiratory and cardiovascular health. Quantum dots (QDs) are ENMs with outstanding semiconductor and fluorescent properties with uses in biomedicine and electronics. QDs are known to induce inflammation and cytotoxicity in rodents and high dose exposures impact lung function 2 weeks after exposure. However, effects of mouse strain and the temporality of QD effects on lung function at more occupationally relevant doses have not been well-established. OBJECTIVE: We evaluated the impact of QD exposure on respiratory mechanics in C57BL/6J and A/J mice. Previous work found a greater initial inflammatory response to QD exposure in A/J mice compared to C57BL/6J mice. Thus, we hypothesized that A/J mice would be more sensitive to QD-induced effects on lung mechanics. METHODS: C57BL/6J and A/J mice were exposed to 6 µg/kg Cd equivalents of amphiphilic polymer-coated Cd/Se core, ZnS shell QDs via oropharyngeal aspiration. Lung mechanics were measured using forced oscillation, and inflammation was characterized by neutrophils and cytokines in bronchoalveolar lavage fluid. RESULTS: Both strains showed signs of QD-induced acute lung inflammation. However, lung mechanics were impacted by QD exposure in A/J mice only. CONCLUSIONS: Our findings suggest that susceptibility to QDs and similar ENM-induced changes in lung function may depend at least in part on genetic background.

Cationic nanoparticle as an inhibitor of cell-free DNA-induced inflammation.

Cell-free DNA (cfDNA) released from damaged or dead cells can activate DNA sensors that exacerbate the pathogenesis of rheumatoid arthritis (RA). Here we show that ~40 nm cationic nanoparticles (cNP) can scavenge cfDNA derived from RA patients and inhibit the activation of primary synovial fluid monocytes and fibroblast-like synoviocytes. Using clinical scoring, micro-CT images, MRI, and histology, we show that intravenous injection of cNP into a CpG-induced mouse model or collagen-induced arthritis rat model can relieve RA symptoms including ankle and tissue swelling, and bone and cartilage damage. This culminates in the manifestation of partial mobility recovery of the treated rats in a rotational cage test. Mechanistic studies on intracellular trafficking and biodistribution of cNP, as well as measurement of cytokine expression in the joints and cfDNA levels in systemic circulation and inflamed joints also correlate with therapeutic outcomes. This work suggests a new direction of nanomedicine in treating inflammatory diseases.

Lipid Stabilized Solid Drug Nanoparticles for Targeted Chemotherapy.

Nanoparticle-based chemotherapeutics have gained widespread interest in medicine due to their tunable pharmacokinetics and pharmacodynamics. Various drug delivery vehicles have been developed including polymer, liposome nanoparticles, and some of them have already made clinical impacts. Despite these advances, drug payload of these formulations is limited (typically <10%). Here, we report a general and scalable approach to prepare lipid-coated solid drug nanoparticles by combining flash nanoprecipitation and extrusion technique, which enables optimization of individual steps separately and flexibility in selection of nanoparticle surface functionalities. Using methotrexate as a model drug, the nanoparticles significantly outperformed free drug in tumor growth suppression.

A universal strategy for the one-pot synthesis of SERS tags.

Surface-enhanced Raman scattering (SERS) tags have attracted tremendous attention in diverse fields owing to their outstanding sensitivity and multiplexing capability. However, the selection of Raman dyes that can be immobilized onto metal nanoparticles is very limited, because certain chemical groups are needed in the dye molecules to interact either with the metal surface or through some intermediate layers. Here, we report a simple, rapid, and robust platform methodology for the one-pot preparation of Raman nanoprobes without the constraints of Raman dye chemical structures. We demonstrate this general approach by immobilizing dye molecules on silver nanoparticle surfaces that were difficult to incorporate previously, and show their applications in multiplexed immunohistochemistry (IHC). We expect that this platform nanotechnology will significantly expand the library of SERS tags and their biomedical uses.

Synthesis of hybrid magneto-plasmonic nanoparticles with potential use in photoacoustic detection of circulating tumor cells.

This article describes a novel synthetic route to obtain hybrid nanostructures that combine the plasmonic properties of gold nanorods with the magnetic properties of iron oxide nanoparticles in a robust silica nanostructure. The silica matrix enhances the physico-chemical stability of the nanostructure and preserves its magneto-plasmonic properties by avoiding the interface between gold and iron oxide. In addition, the magneto-plasmonic features of the nanohybrids can be tuned due to the independent synthesis of each component. The magnetic and plasmonic properties of these nanostructures can potentially enhance the photoacoustic detection of circulating tumor cells. Graphical abstract Schematic presentation of a hybrid magneto-plasmonic nanoparticle with an Au@Fe3O4@SiO2 core-satellite-shell arrangement. The magnetic and plasmonic responses of this kind of nanostructure enable magnetic trapping and photoacoustic detection of circulating tumor cells.

Cross-Platform Cancer Cell Identification Using Telomerase-Specific Spherical Nucleic Acids.

Distinguishing tumor cells from normal cells holds the key to precision diagnosis and effective intervention of cancers. The fundamental difficulties, however, are the heterogeneity of tumor cells and the lack of truly specific and ideally universal cancer biomarkers. Here, we report a concept of tumor cell detection, bypassing the specific genotypic and phenotypic features of different tumor cell types and directly going toward the hallmark of cancer, uncontrollable growth. Combining spherical nucleic acids (SNAs) with exquisitely engineered molecular beacons (SNA beacons, dubbed SNAB technology) is capable of identifying tumor cells from normal cells based on the molecular phenotype of telomerase activity, largely bypassing the heterogeneity problem of cancers. Owing to the cell-entry capability of SNAs, the SNAB probe readily achieves tumor cell detection across multiple platforms, ranging from solution-based assay, to single cell imaging and in vivo solid tumor imaging (unlike PCR that is restricted to cell lysates). We envision the SNAB technology will impact cancer diagnosis, therapeutic response assessment, and image-guided surgery.

Noncovalent tagging of siRNA with steroids for transmembrane delivery.

Short interfering RNA (siRNA) has broad applications in biology and medicine, and holds tremendous potential to become a new class of therapeutics for many diseases. As a highly anionic macrobiomolecule, its cytosolic delivery, however, has been a major roadblock in translation. Here, we report the development of small, bifunctional chemical tags capable of transporting siRNA directly into the cytosol. The bifunctional tags consist of a siRNA-binding moiety that interacts with siRNA non-covalently, and a steroid domain that readily fuses with the mammalian cell membrane. In contrast to the conventional covalently conjugated siRNA-steroid that enters cells largely via endocytosis which substantially limits siRNA bioavailability, the non-covalently tagged siRNA is cell membrane-permeant, avoiding the endocytic pathway. This new methodology enables effective RNA interference (RNAi) without the need of cationic transfection or endosomolytic agents, opening a new avenue for intracellular delivery of native biologics.

Congenital Zika virus infection as a silent pathology with loss of neurogenic output in the fetal brain.

Zika virus (ZIKV) is a flavivirus with teratogenic effects on fetal brain, but the spectrum of ZIKV-induced brain injury is unknown, particularly when ultrasound imaging is normal. In a pregnant pigtail macaque (Macaca nemestrina) model of ZIKV infection, we demonstrate that ZIKV-induced injury to fetal brain is substantial, even in the absence of microcephaly, and may be challenging to detect in a clinical setting. A common and subtle injury pattern was identified, including (i) periventricular T2-hyperintense foci and loss of fetal noncortical brain volume, (ii) injury to the ependymal epithelium with underlying gliosis and (iii) loss of late fetal neuronal progenitor cells in the subventricular zone (temporal cortex) and subgranular zone (dentate gyrus, hippocampus) with dysmorphic granule neuron patterning. Attenuation of fetal neurogenic output demonstrates potentially considerable teratogenic effects of congenital ZIKV infection even without microcephaly. Our findings suggest that all children exposed to ZIKV in utero should receive long-term monitoring for neurocognitive deficits, regardless of head size at birth.

A ribonucleoprotein octamer for targeted siRNA delivery.

Hurdles in cell-specific delivery of small interfering RNA (siRNA) in vivo hinder the clinical translation of RNA interference (RNAi). A fundamental problem concerns conflicting requirements for the design of the delivery vehicles: cationic materials facilitate cargo condensation and endosomolysis, yet hinder in vivo targeting and colloidal stability. Here, we describe a self-assembled, compact (~30 nm) and biocompatible ribonucleoprotein-octamer nanoparticle that achieves endosomal destabilization and targeted delivery. The protein octamer consists of a poly(ethylene glycol) scaffold, a sterically masked endosomolytic peptide and a double-stranded RNA-binding domain, providing a discrete number of siRNA loading sites and a high siRNA payload (>30 wt%), and offering flexibility in both siRNA and targeting-ligand selection. We show that a ribonucleoprotein octamer against the polo-like kinase 1 gene and bearing a ligand that binds to prostate-specific membrane antigen leads to efficient gene silencing in prostate tumour cells in vitro and when intravenously injected in mouse models of prostate cancer. The octamer's versatile nanocarrier design should offer opportunities for the clinical translation of therapies based on intracellularly acting biologics.

Publisher Correction: A ribonucleoprotein octamer for targeted siRNA delivery.

In the version of this Article originally published, in Fig. 3b, middle row, the units 'nM' were incorrect and should have been 'min'. And, in Fig. 4f, in the bottom row, the data in the middle and right panels were mistakenly duplicated from the panels above. These errors have now been corrected.

Dramatic enhancement of the detection limits of bioassays via ultrafast deposition of polydopamine.

The ability to detect biomarkers with ultrahigh sensitivity radically transformed biology and disease diagnosis. However, owing to incompatibilities with infrastructure in current biological and medical laboratories, recent innovations in analytical technology have not received broad adoption. Here, we report a simple, universal 'add-on' technology (dubbed EASE) that can be directly plugged into the routine practices of current research and clinical laboratories and that converts the ordinary sensitivities of common bioassays to extraordinary ones. The assay relies on the bioconjugation capabilities and ultrafast and localized deposition of polydopamine at the target site, which permit a large number of reporter molecules to be captured and lead to detection-sensitivity enhancements exceeding 3 orders of magnitude. The application of EASE in the enzyme-linked-immunosorbent-assay-based detection of the HIV antigen in blood from patients leads to a sensitivity lower than 3 fg ml-1. We also show that EASE allows for the direct visualization, in tissues, of the Zika virus and of low-abundance biomarkers related to neurological diseases and cancer immunotherapy.